Novel findings in patients with primary hyperoxaluria type III and implications for advanced molecular testing strategies

Abstract

Identification of mutations in the HOGA1 gene as the cause of autosomal recessive primary hyperoxaluria (PH) type III has revitalized research in the field of PH and related stone disease. In contrast to the well-characterized entities of PH type I and type II, the pathophysiology and prevalence of type III is largely unknown. In this study, we analyzed a large cohort of subjects previously tested negative for type I/II by complete HOGA1 sequencing. Seven distinct mutations, among them four novel, were found in 15 patients. In patients of non-consanguineous European descent the previously reported c.700+5G>T splice-site mutation was predominant and represents a potential founder mutation, while in consanguineous families private homozygous mutations were identified throughout the gene. Furthermore, we identified a family where a homozygous mutation in HOGA1 (p.P190L) segregated in two siblings with an additional AGXT mutation (p.D201E). The two girls exhibiting triallelic inheritance presented a more severe phenotype than their only mildly affected p.P190L homozygous father. In silico analysis of five mutations reveals that HOGA1 deficiency is causing type III, yet reduced HOGA1 expression or aberrant subcellular protein targeting is unlikely to be the responsible pathomechanism. Our results strongly suggest HOGA1 as a major cause of PH, indicate a greater genetic heterogeneity of hyperoxaluria, and point to a favorable outcome of type III in the context of PH despite incomplete or absent biochemical remission. Multiallelic inheritance could have implications for genetic testing strategies and might represent an unrecognized mechanism for phenotype variability in PH.

Figure 1

(a) HOGA1 mutations identified in patients with PHIII. Electropherograms of the predominant splice-site mutation c.700+5G>T and the four novel HOGA1 point mutations compared with the parental carriers (if available) or wild-type sequence. (b) Pedigree and corresponding electropherograms of kindred 12. Segregation of the c.569C>T (p.P190L) HOGA1 mutation and the c.603C>A (p.D201E) AGXT mutation in a consanguineous kindred from Lebanon.

Figure 2

pSPL3 splicing assay and sequencing result for the frequent splice-site mutation c.700+5G>T compared with wild-type cDNA. Fragments of the human HOGA1 gene containing exon 5 were cloned into the splicing vector pSPL3 generating plasmids pSPL3-HOGA1-Exon5-WT and pSPL3-HOGA1-Exon5+5G>T. After transfection mRNA was extracted from cells and reverse transcribed cDNA was amplified using specific primers within flanking pSPL3 exons. The G>T sequence alteration on position +5 cripples the wild-type donor site and activates an aberrant donor site on position +52, which leads to the in-frame insertion of 51 nucleotides of intron 5 (17 amino acids) into the native protein. The interrupted black lines mark the spliced out intronic sequences. The blue box indicates HOGA1 exon 5 while the blue lines represent the flanking 500 bp of upstream and 900 bp downstream intronic sequence of HOGA1 exon 5. The gray boxes mark pSPL3 exons A and B with the adjacent black lines depicting the flanking intronic sequence of the pSPL3 exons. The red box represents the 51 bp insertion resulting from the c.700+5G>T splice-site mutation.

Figure 3

(a) Subcellular localization of HOGA1 protein in transfected Cos cells. Top panel: confocal microscopy of Cos cells transfected with wild-type human HOGA1 cDNA in pCIneo vector showed the typical filamentous network of mitochondria after incubation with rabbit anti-human HOGA1 antibody and secondary Alexa-488-conjugated anti-rabbit antibody (green fluorescence, left image), which largely coincide with red signal from the mitochondria, visualized using MitoTracker Red (red fluorescence, central image), as can be demonstrated by merging both confocal images (yellow signal, right image). Bottom panel: confocal microscopy of Cos cells transfected with human HOGA1 variants showed mitochondrial subcellular localization after incubation with rabbit anti-human HOGA1 antibody, like above (green fluorescence). The pattern coincided in all cases with MitoTracker labeled mitochondria (data not shown). (b) Western blot of HOGA1 protein in transfected Cos cells. Top panel: 25 μg protein from Cos cells transfected with either wild-type (wt) or mutant HOGA1 cDNAs were electrophoresed in denaturing acrylamide gels, transferred to nitrocellulose and probed with rabbit antibody raised against recombinant human HOGA1. A band slightly above the expected 35.2 kDa of the HOGA1 monomer is observed in all lanes except mutant p.Q116X and untransfected Cos cells (cos). Molecular weight markers of 35 and 42 kDa are shown to the left (arrows). Ten μg mouse liver protein (Lv) were loaded on the right-most lane as a positive control. Bottom panel: loading control showing similar amounts of protein per lane when the filter was re-probed with mouse anti-actin antibody (except for the liver control; last lane to the right, Lv; where the signal could only be seen upon longer exposures of the film (data not shown)).